The aim of the present study was to investigate the effects of exercise during canicross competition on urinary biochemical parameters and urinary proteins. Our results showed that, among the parameters analysed in routine urinalysis, pH was affected by exercise and albuminuria also showed changes immediately after physical exercise with a return to baseline within 2 h after the physical activity. In particular, SDS-PAGE electrophoresis allowed the separation of the urinary proteins on the basis of their molecular mass allowing a qualitative evaluation of the urinary proteome and a quantitative analysis of urine albumin and uromodulin, with a significant increase in urinary albumin and a decrease in uromodulin after exercise. In this study, urine was collected by spontaneous voiding and the use of this non-invasive sampling method proved useful in obtaining information on the health of the animals in the immediate moments before and after the competition without increasing the stress of the animals involved in the competition by any visits or invasive manipulations by veterinarians.